Enterococci are among the most important pathogens associated with infections in hospitalized patients. Especially the presence of multiple antibiotic resistance determinants in most clinically relevant isolates urges the development of alternative treatment and prevention strategies to combat these sometimes untreatable infections. Enterococci have developed specific mechanisms to acquire and transmit DNA horizontally, and these traits are responsible for numerous outbreaks in the hospital setting.
At least 15 enterococcal species exist, but only two of them are commonly associated with clinical infection, namely, Enterococcus faecalis, which is responsible for 80% infections caused by enterococci, and Enterococcus faecium. The Gram-positive bacterium Enterococcus faecalis is a natural inhabitant of the mammalian gastrointestinal tract, and is commonly found in soil, sewage, water, and food, frequently through faecal contamination (Klare, I., Werner, G. and Witte, W. Contrib. Microbiol. 2001, 8, 108-22).
Sensitive strains of these bacteria can be treated with ampicillin and vancomycin. Nevertheless, some enterococci are resistant to β-lactam-based antibiotics (intrinsically against all cephalosporins and in some cases acquired against penicillins through a beta-lactamase) as well as aminoglycosides (intrinsic low-level resistance, acquired high-level resistance). In the last two decades, particularly virulent strains of Enterococcus that are resistant to vancomycin (Vancomycin-resistant Enterococcus, or VRE) have emerged in nosocomial infections of hospitalized patients especially in the US, but to a lesser extent also in countries all over the world. The increasing occurrence of enterococcal strains resistant to multiple antibiotics underscores the necessity to improve the understanding of the pathogenesis of infection (Murray, B. E. N. Engl. J. Med. 2000, 342, 710-721; Theilacker, C., Krueger, W. A., Kropec, A. and Huebner, J. Vaccine 2004, 22 Suppl 1, S31-8).
Teichoic acids can be found in the cell wall of gram-positive bacteria, such as Staphylococci, Streptococci, Bacillus, Clostridium, Corynebacterium and Listeria, and appear to protrude from the surface of the peptidoglycan layer. Teichoic acids are not found in gram-negative bacteria. They can be covalently linked to N-acetylmuramic acid of the peptidoglycan layer (wall teichoic acid, WTA), or attached via a lipid anchor in the cytoplasmic membrane (lipoteichoic acid, LTA).
The main function of teichoic acids is to provide rigidity to the cell-wall by attracting cations such as magnesium and sodium. Teichoic acids are usually, but not always, substituted with D-alanine ester residues, giving the molecule zwitterionic properties. These zwitterionic teichoic acids are suspected ligands for toll-like receptors 2 and 4. Teichoic acids also assist in regulation of cell growth by limiting the ability of autolysins to break the β(1-4) bond between the N-acetyl glucosamine and the N-acetylmuramic acid. Teichoic acids serve as an attachment site for some parasites. Destruction of the bacteria and release of the teichoic acid into the bloodstream may cause fever, blood vessel dilation and possibly shock and subsequent death. Teichoic acid can also be used by bacteria to attach to mucosal membranes.
Enterococcal LTA is a complex molecule consisting of a glycolipid anchor and a polyglycerol phosphate chain substituted non-stochiometrically with alanine, kojibiose, and alaninated kojibiose (Theilacker, Kaczynski et al. 2006). LTA is an integral component of the gram-positive cell wall, and antibodies against this antigen have been shown to be protective against enterococcal infections (Huebner, Wang et al. 1999; Huebner, Quaas et al. 2000; Theilacker, Kaczynski et al. 2006). However, the antigenic component constituting the protective epitope has been not defined so far. Structurally similar molecules exist in other clinically relevant gram-positive bacteria (such as streptococci, staphylococci, listeria, etc.) and rabbit immune sera raised against an Enterococus faecalis LTA has been shown to be protective also against Streptococcus agalactiae and Staphylococcus aureus (Huebner at el., unpublished observation).
Teichoic acids and lipoteichoic acids have been considered strong exogenous pyrogens, i.e. they belong to the substances that may lead to a feverish reaction in a human after a bacterial infection by gram-positive bacteria. They are most likely recognized by the toll-like receptor TLR-2 that is expressed on monocytes and dendritic cells, B- and T-lymphocytes and macro-phages. Furthermore, they lead to the excretion of cytokines, and therefore are one factor for the inflammatory reaction following such an infection.
Due to their antigenic properties, they have also been proposed as interesting candidates for the development of synthetic vaccines.
U.S. Pat. No. 7,011,826 describes a vaccine for the prevention of lactic acidosis in a vertebrate, said vaccine comprising at least one isolated microorganism, or fragment or fragments thereof, wherein said microorganism is capable of producing lactic acid within the gut of said vertebrate, and wherein said microorganism is selected from the group consisting of: Clostridium-like species, Prevotella-like species, Bacteroides-like species, Enterococcus-like species, and Selenomonas species.
Using structural information derived from above-mentioned studies, Hogendorf and colleagues recently synthesized mimetics of the Enterococcus glycerophosphate backbone (Hogendorf, Bos et al. 2010).
Morath et al (in Morath et al. Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release. J Exp Med. Jun. 17, 2002; 195(12):1635-40.) describe the synthesis of a complete LTA molecule with six glycerophosphate units carrying four alanine plus one N-acetyl-glucosamine substituent, which displayed the same potency to activate monocytes as native LTA. However, 100-1,000 times higher concentrations of the lipid anchor were required for cytokine induction. Of note, this synthetic molecule was not developed as a therapeutic agents but to prove the hypothesis that LTA has pro-inflammatory activity. Furthermore, the complexity of the synthetic steps involved in producing their synthetic LTA precludes the use of this material as a vaccine.
Wang et al (in Wang Y, Huebner J, Tzianabos A O, Martirosian G, Kasper D L, Pier G B. Structure of an antigenic teichoic acid shared by clinical isolates of Enterococcus faecalis and vancomycin-resistant Enterococcus faecium. Carbohydr Res. Mar. 31, 1999; 316(1-4):155-60) describe a shared antigenic teichoic acid, previously found to be a surface capsule-like poly-saccharide, from clinical isolates of Enterococcus faecalis and vancomycin-resistant E. faecium. It was composed of glucose, glycerol, and phosphate as determined by chemical and GC-MS analysis. The repeating-unit structure was elucidated by a series of 1H, 13C, and 31P NMR spectroscopy.
Deininger S et al. (in Deininger et al., Use of synthetic derivatives to determine the minimal active structure of cytokine-inducing lipoteichoic acid. Clin Vaccine Immunol. December 2007; 14(12):1629-33. Epub Oct. 10, 2007) describe that the chemical synthesis of LTA proved its immunostimulatory properties. To determine the minimal active structure of LTA, they reduced synthetic LTA in a number of steps down to the synthetic anchor and employed these molecules to stimulate interleukin-8 (IL-8) release in human whole blood. Ten times more of the synthetic structures with four to six d-alanine-substituted polyglycerophosphate units (50 nM) than of the native LTA preparation was required to induce IL-8 release. A further reduction to three backbone units with two or no d-alanine residues resulted in cytokine induction only from 500 nM. When the LTA derivatives were used at 500 nM, they induced increasing levels of IL-8 and tumor necrosis factor alpha with increasing elongation of the backbone. TLR2 dependency could be shown only with cells from TLR2-deficient mice for the two largest synthetic structures. This was confirmed by using TLR2-transfected HEK 293 cells. Taken together, these data indicate that although the synthetic anchor (which, unlike the native anchor, contains only myristic acid) cannot induce cytokine release, the addition of three backbone units, even without d-alanine substituents, confers this ability. Lengthening of the chain with d-alanine-substituted backbone units resulted in increased cytokine-inducing potency and a more sensitive response.
In the elucidation of the molecular mode of action of LTAs in effecting an immune response, pure and well-defined fragments would be valuable tools. Furthermore, an effective synthesis would also allow to produce sufficient amounts of material as needed. It is therefore an object of the present invention to provide pure and well-defined effective synthetic fragments (mimetics) of LTA, in particular in order to develop a new promising vaccine for an active or passive immunotherapy of bacteria, and in particular enterococci. Furthermore, improved methods for an automated synthesis shall be provided.
The present invention fulfils these needs by providing new synthetic lipoteichoic acid (LTA) mimetic having the following general formula (I)

wherein R1 is selected from H or a carbohydrate moiety, such as, for example, a glucosyl moiety (Glc), L is a linker moiety for connecting said mimetic to a suitable carrier protein, and n is an integer selected from 2 to 40, preferably selected from 4 to 6, or salts or solvates thereof.
This synthetic LTA cell wall mimetic provides a new and effective antigenic target for the development of more efficient strategies to effectively treat and/or prevent infection in vertebrates caused, at least in part, by enterococci or other gram-positive bacteria, allows for improved vaccination strategies, and allows the development and production of respective vaccines, such as glycoconjugate vaccines.
The inventors set out to develop a synthetic methodology to efficiently assemble LTA types of glycopolymers. In this respect it is of interest to note that structure and activity studies of synthetic derivatives of Staphylococcus aureus lipoteichoic acid (LTA) fragments led to the discovery of ligands for the human TLR-2 receptor (see, for example, Morath et al. and Deininger S et al. as cited above). The inventors directed their first attention at the TA of Enterococcus faecalis, which is build up from 1,3-poly(glycerolphosphate) randomly decorated at the C-2 positions with D-alanine, kojibio se (α-D-glucopyranosyl-(1→2)-α-D-glucose), or 6,6′-di-alanyl-α-kojibiose.
The above compounds include a linker group L in order to be coupled or conjugated to other chemical entities. These linker groups are known in the state of the art, and usually are immunologically inactive, i.e. do not interfere with the immunological properties of the synthetic LTA mimetic. Preferred linkers include, but are not limited to, C1 to C12 alkylamino linkers, which are optionally substituted with other groups, or peptide linkers. Other modifications include the addition of chemical moieties to the LTA mimetic (also included in “L”) in order to carry a detectable label, such as chelating groups or enzymatic groups. Furthermore, peptide (e.g. His) or other “labels” or “tags” can be added in order to be able to purify and/or use the synthetic LTA mimetic, for example in diagnostic assays. Thus, preferred is a synthetic lipoteichoic acid (LTA) mimetic according to the present invention, wherein L is selected from an alkylamino group having the general formula (II)

wherein m is an integer selected from 1 to 20.
The synthetic lipoteichoic acid (LTA) mimetic according to the present invention furthermore include carbohydrate moieties, such as, for example, a glucosyl (Glc) moiety. Preferably, these carbohydrate moieties units are selected from monosaccharide or disaccharide units as found in enterococci or staphylococci, such as glucose (Glu), glucosamine, N-acetyl glucosamine, galactose, rhamnose, gentiobiose, or kojibiose.
Further preferred is a synthetic lipoteichoic acid (LTA) mimetic according to the present invention, having the following general formula (III)

wherein z is an integer selected from 1 to 40, and preferably selected from 4 to 6.
Also further preferred is a synthetic lipoteichoic acid (LTA) mimetic according to the present invention, having the following general formula (IV)

wherein z is an integer selected from 1 to 40, and preferably selected from 4 to 6.
In both these embodiments, L preferably is
Nevertheless, of course this group can also be used with the other synthetic lipoteichoic acid (LTA) mimetics of the present invention. Further preferred are compounds WH6 and WH7 as described herein (see Figures).
In general, the synthetic lipoteichoic acid (LTA) mimetic according to the present invention includes 2 to 40 repeating polyglycerol phosphate units, such as, for example, 3, 4, 5, or 6, or 10 to 20. Although it is speculated that the immunologic activity of said molecule increases with its length of polyglycerol phosphate units, the inventors could show that shorter molecules can already be quite effective in their immune response (such as, for example, hexamers, see examples). Due to the fact that these molecules are easier to synthesize, a molecule wherein n or z is 5 or 6 is most preferred.
Another aspect of the invention then relates to a pharmaceutical composition, comprising at least one of the synthetic lipoteichoic acid (LTA) mimetic according to the present invention and/or at least one antibody according to the present invention as described below, together with at least one pharmaceutically acceptable carrier, adjuvant and/or diluent.
Particularly preferred is a pharmaceutical composition according to the present invention, wherein said composition comprises a synthetic lipoteichoic acid (LTA) mimetic as described herein.
Further preferred is a pharmaceutical composition according to the present invention, wherein said composition is formulated as a vaccine, in particular against infections caused by enterococci, in particular antibiotic resistant enterococci, such as VRE strains, preferably of E. faecalis or E. faecium. Most preferred is a pharmaceutical composition according to the present invention, wherein said synthetic lipoteichoic acid (LTA) mimetic according to the present invention is present in a glycoconjugate vaccine.
The synthetic lipoteichoic acid (LTA) mimetic according to the present invention (either present as the antigen alone or in a bacterial extract or cell wall fraction) is preferably used for an enterococcal, staphylococcal, or pneumococcal vaccine, either for active or passive immunization.
Thus the invention further provides a pharmaceutical composition, and in particular a vaccine, for the prevention of enterococcal infections in a vertebrate, said pharmaceutical composition comprising at least one synthetic lipoteichoic acid (LTA) mimetic according to the present invention, optionally together with a pharmaceutically acceptable carrier, adjuvants and/or diluent. Preferred carriers include, but are not limited to, CRM (CRM197), Tachypleus tridentatus hemocyanin (TTH), Limulus polyphemus hemocyanin (LPH), tetanus toxoid (TT), diphtheria toxoid (DT), bovine serum albumin (BSA), and the ExoU protein.
Typically, the vaccine can further comprise live or dead intact cells of at least one enterococcal strain, preferably of E. faecalis, together with the synthetic lipoteichoic acid (LTA) mimetic of the invention. More typically, the vaccine comprises cell lysate from at least one enterococcal strain. Most preferred is a glycoconjugate vaccine comprising the synthetic lipoteichoic acid (LTA) mimetic according to the present invention. The methods for purifying the selected bacterial fractions containing enterococcal antigens are known to the person of skill. Another aspect relates to a pharmaceutical composition or vaccine, wherein the synthetic lipoteichoic acid (LTA) mimetic as included has been produced, at least in part, through chemical synthesis.
Typically, the vertebrate is a monogastric, herbivore or ruminant animal or human subject. Even more typically, the vertebrate is selected from the group consisting of human, non-human primate, murine, bovine, ovine, equine, porcine, caprine, leporine, avian, feline and canine. More typically, the vertebrate is selected from the group consisting of human, ovine, camelids, porcine, bovine, equine or canine.
The pharmaceutical composition can be formulated for administration via intramuscular, sub-cutaneous, topical or other parenteral route. In general, the microorganisms of the present invention are commensal in nature. Thus, oral administration is generally not an effective route of vaccination, and as a consequence, administration via an intramuscular, subcutaneous topical or other parenteral route is preferred. Preferably, the vaccine is formulated for administration via intramuscular, subcutaneous, or inhalation routes. The vaccine may also include cytokines, such as: G-CSF, GM-CSF, interleukins or tumor necrosis factor alpha, used singly or in combination.
The pharmaceutical composition may also include an adjuvant. More typically, the adjuvant is selected from the group consisting of Freunds Complete/Incomplete Adjuvant, Montenide Macrol Adjuvant, Phosphate Buffered Saline and Mannan oil emulsions, saponins (QuiLA) dextran (dextran sulphate, DEAE-Dextran), aluminum compounds (Imject Alum), N-acetylglucosamiyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (Gerbu adjuvant). More typically, the adjuvant is selected from the group as described in the Vaccine 1995, vol 13, page 1203; 1993 vol 11 page 293; and 1992 vol 10 page 427, the disclosures of which are incorporated herein by reference.
Yet another important aspect of the present invention then relates to the synthetic lipoteichoic acid (LTA) mimetic according to the present invention, the antibody according to the present invention, or the pharmaceutical composition according to the present invention for use in the treatment of diseases, such as bacterial infections, in particular by a Gram-positive bacterium, such as, for example, bacterial infection, enterococcal infection, urinary tract infections, bacteremia, endocarditis, bacterial endocarditis, peritonitis, wound and soft tissue infections, and meningitis, or pneumonia, and foreign body infections. Said Gram-positive bacterium can be preferably selected from enterococci, staphylococci or streptococci, such as, for example, E. faecium, E. faecalis, S. aureus, coagulase-negative staphylococci or S. pyogenes, and in particular antibiotic-resistant strains thereof.
Another aspect of the invention then relates to a method for producing the synthetic lipoteichoic acid (LTA) mimetic of the invention, wherein said method comprises synthesizing said mimetic through chemical synthesis, comprising, for example, solution phase and/or solid phase chemistry. In the context of the experiments of the present invention, kojibiosyl-glycerol phosphoramidite in combination with a glycerolphosphoramidite, an aminohexyl-phosphoramidite and dibenzylglycerol were coupled to a fully protected glycerol TA hexamer, using chemistry that can be amended for automated synthesis. Global deprotection afforded the desired hexamer.
Preferred is a method for producing the synthetic lipoteichoic acid (LTA) mimetic according to the present invention, comprising synthesizing said molecule on a solid phase material. The methods of the present invention more preferably are in an automated solid-phase synthesis format. Such a format has been developed by the inventors and used to construct WH6 (FIG. 1) and WH7 (FIG. 2), employing commercially available automated DNA/RNA synthesis equipment in combination with tailor made glycerol phosphoramidite building blocks.
Another aspect of the invention relates to an antibody, preferably a monoclonal antibody or antigenic fragment thereof, that specifically recognizes the synthetic lipoteichoic acid (LTA) mimetic according to the present invention. The term “antibody” shall include both monoclonal or polyclonal antibodies, recombinant antibodies or fragments thereof, such as Fab and the like, as well as human or humanized antibodies.
Another aspect of the invention then relates to a method for producing the antibody according to the present invention, comprising immunizing a mammal, preferably a rabbit, with the synthetic lipoteichoic acid (LTA) mimetic according to the present invention, or a with the pharmaceutical composition according to the present invention, or preferably the vaccine according to the present invention, and, optionally isolating said antibody from said animal. Respective methods are known to the person of skill, and are disclosed in the state of the art.
Yet another aspect of the present invention then relates to a method for producing a monoclonal antibody according to the present invention that is specific for the synthetic lipoteichoic acid (LTA) mimetic according to the present invention, comprising generating hybridoma cells producing said antibody as a monoclonal antibody, or comprising a recombinant production of said antibody in a host cell. Respective methods are known to the person of skill, and are described in the state of the art.
Still another important aspect of the present invention then relates to the use of the synthetic lipoteichoic acid (LTA) mimetic according to the present invention as an antigen in the production of antibodies that are specific for said antigen.
Yet another important aspect of the present invention then relates to the use of the synthetic lipoteichoic acid (LTA) mimetic according to the present invention, the antibody according to the present invention, or the pharmaceutical composition according to the present invention for the treatment against bacterial infections or for the production of a medicament for the prophylactic or therapeutic treatment of a disease or condition caused by bacterial infections, in particular enterococcal infection, such as nosocomial infection, bacteraemia, endocarditis, urinary tract infections, surgical wound infections, peritonitis, wound and soft tissue infections, meningitis, pneumonia, and foreign body infections, in particular caused by antibiotic resistant enterococci, such as VRE strains, such as E. faecalis, and also staphylococci and streptococci. Preferably, said medicament is a vaccine as described herein.
According to yet another preferred embodiment of the invention, there is provided a method for inducing an immune response against at least one Gram-positive bacterial strain, such as an enterococcal strain, comprising the synthetic lipoteichoic acid (LTA) mimetic of the present invention in a vertebrate, said method comprising administering to said vertebrate an immunologically effective amount of the vaccine in accordance with the invention, or a pharmaceutical composition in accordance with the invention.
According to yet another preferred embodiment of the invention, there is provided a method for treating or preventing a bacterial infection in a vertebrate, comprising administering to said vertebrate a therapeutically effective amount of the synthetic lipoteichoic acid (LTA) mimetic according the present invention, the antibody according to the present invention, or the pharmaceutical composition according to the present invention.
Preferred is a method according to the present invention, wherein said bacterial infection, in particular enterococcal infection, is a nosocomial infection, bacteraemia, endocarditis, urinary tract infections, surgical wound infections, peritonitis, wound and soft tissue infections, meningitis, pneumonia, or foreign body infections, in particular caused by antibiotic resistant enterococci, such as VRE strains, such as E. faecalis, Staphylococci or Streptococci. 